Polynucleotide encoding a novel human nm23-like protein

ABSTRACT

The present invention provides polynucleotides which identify and encode a novel human nm23-like protein (H-nm23). The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding H-nm23 and for a method for producing the protein. The invention also provides for the use of substantially purified H-nm23 for the treatment of diseases associated with the expression of H-nm23. The invention also describes diagnostic assays which utilize diagnostic compositions comprising the polynucleotides which hybridize with naturally occurring sequences encoding H-nm23 and antibodies which specifically bind to the protein.

This application is a divisional application of U.S. application Ser.No. 08/713,825, filed Sep. 13, 1996, now U.S. Pat. No. 5,874,285.

FIELD OF THE INVENTION

The present invention relates to nucleic acid and amino acid sequences anovel human nm23-like protein and to the use of these sequences in thediagnosis, study, prevention and treatment of disease.

BACKGROUND OF THE INVENTION

The nm23 genes encode proteins that participate in the development anddifferentiation of normal tissues. Nm23 proteins are also associatedwith the regulation of tumor metastasis. Homologs of the highlyconserved protein have been characterized in numerous tissues includingthose from humans, mice, Drosophila, and Myxococcus xanthus. The nm23proteins generally consist of 150 to 180 amino acid residues. All knownnm23 proteins contain a leucine zipper motif and exhibit nucleosidediphosphate kinase (NDPK) activity.

Nm23 protein accumulation is coincident with the functionaldifferentiation of multiple epithelial tissues in the developing mouse.At the onset of organogenesis, the amount of nm23 protein is relativelylow and uniform throughout the mouse embryo. The protein begins toaccumulate preferentially in the first embryonic tissues todifferentiate, the developing nervous system and heart. Subsequentdifferentiation of liver, kidney, skin, intestine, adrenal, and stomachepithelial cells is accompanied by increased nm23 protein expression(Lasko M et al (1992) Cell Growth Differ 3:873-879).

In rodent, reduced expression of the nm23 gene systems has beencorrelated with increased potential for tumor metastasis. A suppressiveeffect of nm23 on several aspects of the cancer process, includingmetastasis, has been demonstrated in murine melanoma cells (Leone A etal (1991) Cell 65: 25-35). The number of metastases developed in amurine melanoma subline was inversely correlated with the expression oftwo murine isotypes, nm23-M1 and nm23-M2 (Baba H et al (1995) Cancer Res55:1977-1981).

In Drosophila, an nm23 homologue, abnormal wing discs (awd), isessential for normal development. Mutation or reduced expression of awdcauses abnormal tissue morphology, necrosis and widespread aberrantdifferentiation similar to malignant progression (Rosengard AM et al(1989) Nature 342:177-180).

Two human nm23 soforms, nm23-H1 and nm23-H2, each consist of 152 aminoacid residues with Mr of 17,143 and 17,294, respectively. The isoformshave 88% sequence identity and encode polypeptides identical to the Aand B chains of human erythrocyte NDPK (Gilles et al (1991) J Biol Chem266:8784-8789). NDPK is a hexameric enzyme, with isozymes consisting ofall combinations of the A and B chains (A6, A5B . . . AB5, B6). NDPKtransfers a phosphoryl group between nucleoside tri- and diphosphatesvia a covalent phosphoenzyme intermediate. In nm23-H1 and -H2, histidine118 is the site of this transient phosphorylation.

Human nm23-H2 protein is also identical to the c-myc purine-bindingtranscription factor PuF (Postel EH et al (1993) Science 261:478-480).Myc, the protein product of the c-myc proto-oncogene, is proposed tomodulate the expression of genes involved in cellular proliferation,differentiation, and tumor formation. Native PuF and purifiedrecombinant nm23-H2 bind to DNA sequences corresponding to anuclease-hypersensitive element (NHE) in the human c-myc P1 promoter,and induce accurate c-myc transcription in vitro.

The relationship between the DNA binding, transcriptional activation,and NDPK activities of nm23 was assessed by site-directed mutagenesis ofrecombinant nm23-H2. Although the NDPK phosphoenzyme active site mutantH118F was inactive in NDPK assays, it displayed normal DNA bindingaffinity for the c-myc promoter and retained full c-myc transcriptionalactivity in vitro (Postel E H and Ferrone C A (1994) J Biol Chem269:8627-8630). This suggests that the DNA binding/transcriptionalactivation and the NDPK activities of nm23-H2 are independent propertiesof the nm23 proteins which may be associated with different biologicalfunctions.

The mechanism by which nm23 affect metastasis and development isunclear. Autophosphorylation of serine has been observed in nm23,distinct from NDPK-associated histidine phosphorylation (MacDonald N Jet al (1993) J Biol Chem 268:25780-25789). A direct correlation has beenobserved in mice between in vivo nm23 serine phosphorylation levels andsuppression of tumor metastatic potential among control and nm23-M1transfected murine melanoma cells. The serine phosphorylation of mousenm23 is inhibited by cAMP in vitro and forskolin in vivo, suggestingthat this phosphorylation is regulated by a signal transduction pathway.No correlation was found between nm23 NDPK activity and melanoma cellmetastasis, nor was NDPK activity inhibited by cAMP (MacDonald, supra).

Co-regulated expression of nm23 and mts1 (cyclin-dependent proteinkinase), another tumor-suppressor gene, alters the state of tubulinpolmerization in B16 melanoma cell lines (Lakshmi M S et al (1993)Anticancer Res 13:299-303). The altered tubulin polymerization issuggested to impart invasive and metastasizing properties to the cellline.

Recently, a new human nm23 isoform, DR-nm23, has been cloned from achronic myelogenous leukemia (CML) blast crisis cell line (Venturelli Det al (1995) Proc Natl Acad Sci USA 92:7435-7439). The DR-nm23 proteinconsists of 168 amino acids, and has 67% and 69% sequence identity tothe nm23-H1 and H2 isoforms, respectively. DR-nm23 is involved in normalhematopoiesis and, when overexpressed, may contribute to differentiationarrest, a feature of blastic transformation in CML.

The NM23 Gene Family and Cancer

Expression levels of nm23 have been monitored throughout the developmentand metastasis of several types of cancer. In some tumors types, aninverse correlation exists between expression of nm23 and metastasis.For instance, elevated nm23 expression in human breast cancer tumors isassociated with a decrease in lymph node metastasis and with longerpatient survival. The nm23 gene product may play an important role insuppressing the metastatic phenotype (Hennessy et al (1991) J NatlCancer Inst 83: 281-285). Stahl et al ((1991) Cancer Res 51:445-449)report that metastatic breast tumors exhibit significantly reducedlevels of the nm23-H1 protein relative to the nm23-H2 protein. TokunagaY et al ((1993) Int J Cancer 55:66-71) likewise report that expressionof nm23-H1, but not nm23-H2, is inversely associated with lymph-nodemetastasis. Overall survival is better in patients in which nm23-H1expression is elevated than in those in which it is lowered. Nm23-H1therefore has value for predicting long-term survival of humanbreast-cancer patients.

Steeg P S et al ((1993) Breast Cancer Res Treat 25:175-187) report asignificant association between reduced nm23 expression, at the RNA orprotein levels, and aggressive tumor growth in human breast cancer.Decreases in nm23 expression begin prior to actual histologicallyidentifiable invasion. Expression of human nm23-H1 cDNA in thetransfected metastatic human MDA-MB-435 breast carcinoma cell linesuppresses -netastatic potential by 50-90%. Transfection of human breastcarcinoma cell lines with a nm23-H1 expression vector restores manyphenotypically normal features to these cells (Howlett A R et al 1994) JNatl Cancer inst 86: 1838-1844).

Differences in expression levels of nm23-H1 have been reported amongnormal ovary tissue, benign tumors and carcinomas. The nm23-H1 proteinis absent from metastatic ovarian carcinomas more often thannon-metastatic carcinomas (Kapitanovic S et al (1995) Anticancer Res1555:587-590). Tumors that do not metastasize into the lymph nodesexpress nm23-H1 at significantly higher levels than tumors that dometastasize into the lymph nodes. These observations suggest that thenm23-H1 protein may have an inhibitory effect on lymphatic metastasis(Viel A et al (1995) Cancer Res 55:2645-2650).

Low levels of nm23 mRNA also correlate with high metastatic potential inmalignant melanomas (Florenes V A et al (1992) Cancer Res 52: 6088-91).However, patients with higher nm23 expression in metastatic tissue tendto live longer (Xerri L et al (1994) Br J Cancer 70:1224-1228).

Similar correlations between nm23 and metastasis are reported forhepatocellular carcinomas (Iizuka et al. 95), prostate cancer (Fishmanet al. 94), and leukemia cell lines (Yamashiro et al. 94).

However, among other tumor types, nm23 expression has no apparentrelationship to metastatic potential. Expression of nm23 even correlatesdirectly with severity in some of these cancers. For instance, nm23expression in various types of thyroid cancers does not appear to play arole in metastasis. The average level of nm23 gene expression in stagesI through III of differentiated thyroid carcinoma is comparable to thatin multinodular goiters. In advanced stages of thyroid carcinoma (stageIV and anaplastic), a 2-fold increase in nm23 expression is observed.This suggests a direct correlation of nm23 expression with rapid cellproliferation in thyroid cancer (Zou M et al (1993) Br J Cancer68:385-388).

Yamaguchi A et al ((1993) Br J Cancer 68:1020-1024) report that nosignificant correlation exists between nm23-H1 expression and colorectaltumor histology, serosal invasion, lymphatic invasion, venous invasion,or lymph node metastasis. However, Zeng Z S et al ((1994) Br J Cancer70:1025-1030) report that nm23-H1 expression increases with localcolorectal tumor severity, and in liver metastases even higher levels ofnm23-H1 expression are found. In addition, Zeng et al (supra) observedtwo immunoreactive species of nm23-H1 expressed in roughly equalproportions in 16 patients: the predicted 17 kDa form and a larger 18.5kDa form.

High expression levels of a 19 kDa form of nm23-H1 protein inneuroblastoma are associated with advanced stage (III and IV) diseaseand with N-myc gene amplification (Hailat N et al (1991) J Clin Invest88: 341-345).

Engel M et al ((1993) Int J Cancer 55:375-9) show that high levels ofnm23-H1 and nm23-H2 mRNA in human squamous-cell lung carcinoma,large-cell carcinoma, sarcoma, and carcinoids are associated with poordifferentiation, advanced stage tumors and poor prognosis. In humanrenal carcinoma cell lines and in high stage renal cancers, levels ofnm23-H1 and nm23-H2 mRNAs were elevated (Kanayama H et al (1994) Int JUrol 1: 324-331).

The discovery of DR-nm23 (Venturelli D et al, supra) raises thepossibility that additional nm23-like proteins present in human tissuesmediate normal or abnormal cellular processes within those tissues.Along with its diagnostic and therapeutic value, the nm23 proteins havea pronounced prognostic value for those types of tumors in which arelationship between expression of nm23 and metastasis exist. Nm23 isalso useful in diagnostic, prognostic and therapeutic applications forthose tumor types for which levels of expression correlate with tumorseverity. Therefore, the selective modulation of the expression oractivity of a novel nm23-like protein may allow the successful treatmentof amenable types of cancer.

SUMMARY OF THE INVENTION

The present invention discloses a novel nm23-like protein, hereinafterreferred to as H-nm23, having chemical and structural homology to humannm23 isoforms DR-nm23, nm23-H1 and nm23-H2. Accordingly, the inventionfeatures a substantially purified H-nm23, encoded by amino acid sequenceof SEQ ID NO:1, having structural characteristics of the family of nm23proteins.

One aspect of the invention features isolated and substantially purifiedpolynucleotides which encode H-nm23. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2. In addition,the invention features nucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention further relates to nucleic acid sequence encoding H-nm23,oligonucleotides, peptide nucleic acids (PNA), fragments, portions orantisense molecules thereof. The present invention also relates to anexpression vector which includes polynucleotide encoding H-nm23 and itsuse to transform host cells or organisms. The invention also relates toantibodies which bind specifically to the nm23 having amino acidsequence of SEQ ID NO:1 and to a pharmaceutical composition comprising asubstantially purified nm23 having amino acid sequence of SEQ ID NO:1.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A through 1C show the amino acid sequence (SEQ ID NO:1) and thenucleic acid sequence (SEQ ID NO:2) of the human nm23-like proteinH-nm23. The alignment was produced using MacDNAsis software (HitachiSoftware Engineering Co Ltd, San Bruno Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among H-nm23(SEQ ID NO:1), the human nm23 isoforms DR-nm23 (GI 1051256; SEQ IDNO:3), nm23-H1 (GI 468542; SEQ ID NO:4), and nm23-H2 (GI 127983, SEQ IDNO:5). The multisequence alignment program of DNAStar software (DNAStarInc, Madison Wis.) was used.

FIG. 3 shows the hydrophobicity plot (generated using MacDNAsissoftware) for H-nm23, SEQ ID NO:1; the X axis reflects amino acidposition, and the negative Y axis, hydrophobicity.

FIGS. 4A and 4B list Incyte libraries in which full-length or partialH-nm23 mRNA sequences are represented, the abundance of the sequences,and the percent abundance.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,amino acid sequence as used herein refers to peptide or proteinsequence.

"Consensus" as used herein may refer to a nucleic acid sequence 1) whichhas been resequenced to resolve uncalled bases, 2) which has beenextended using XL-PCR (Perkin Elmer) in the 5' or the 3' direction andresequenced, 3) which has been assembled from the overlapping sequencesof more than one Incyte clone GCG Fragment Assembly System, (GCG,Madison Wis.), or 4) which has been both extended and assembled.

"Peptide nucleic acid" as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen P E et al(1993) Anticancer Drug Des 8:53-63).

A "variant" of H-nm23 is defined as an amino acid sequence that isdifferent by one or more amino acid substitutions. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, eg, replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,eg, replacement of a glycine with a tryptophan. Similar minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNAStar software.

A "deletion" is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring H-nm23.

A "substitution" results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

The term "biologically active" refers to a H-nm23 having structural,regulatory or biochemical functions of the naturally occurring H-nm23.Likewise, "immunologically active" defines the capability of thenatural, recombinant or synthetic H-nm23, or any oligopeptide thereof,to induce a specific immune response in appropriate animals or cells andto bind with specific antibodies.

The term "derivative" as used herein refers to the chemical modificationof a nucleic acid encoding H-nm23 or the encoded H-nm23. Illustrative ofsuch modifications would be replacement of hydrogen by an alkyl, acyl,or amino group. A nucleic acid derivative would encode a polypeptidewhich retains essential biological characteristics of natural H-nm23.

As used herein, the term "substantially purified" refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

"Stringency" typically occurs in a range from about Tm-5° C. (5° C.below the Tm of the probe) to about 20° C. to 25° C. below Tm. As willbe understood by those of skill in the art, a stringency hybridizationcan be used to identify or detect identical polynucleotide sequences orto identify or detect similar or related polynucleotide sequences.

The term "hybridization" as used herein shall include "any process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing" (Coombs J (1994) Dictionary of Biotechnoloay, StocktonPress, New York N.Y.). Amplification as carried out in the polymerasechain reaction technologies is described in Dieffenbach C W and G SDveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.).

Preferred Embodiment

The present invention relates to a novel human nm23-like protein,designated H-nm23, initially identified among the partial cDNAs from ahuman breast tissue library (BRSTNOT05) and to the use of the nucleicacid and amino acid sequences disclosed herein in the study, diagnosis,prevention and treatment of disease. Northern analysis using theLIFESEQ™ database (Incyte Pharmaceuticals, Palo Alto Calif.) shows thatmRNA encoding H-nm23 was found in tissues isolated from small intestine,prostate, ovary, lymph node, breast spinal cord, thyroid, colon, lungand bladder tissues (FIG. 4). Of the 41 cDNA libraries containing H-nm23mRNA, ten were derived from prostate tissue. Five of these librarieswere from prostate rumors. Of the five non-tumor prostate libraries,four were generated from non-cancerous tissues excised from cancerpatients, and the remaining library was derived from a young manasympomatic of cancer. H-nm23 mRNA was also found in fetal and neoatalheart, spleen, lung, and kidney, but was not found in these organs inadults, indicating that H-nm23 may be related to the development ofthese tissues. The single exception to this trend was the presence ofH-nm23 in adult heart myoma (atrial myxoma), a benign tumor. H-nm23 mRNAwas also transcribed in the hNT2 cell line, which was derived from ahuman teratocarcinoma that exhibited properties characteristic of acommitted neuronal precursor at an early stage of development.

The present invention also encompasses H-nm23 variants. A preferredH-nm23 variant is one having at least 80% amino acid sequence similarityto the H-nm23 amino acid sequence (SEQ ID NO:1), a more preferred H-nm23variant is one having at least 90% amino acid sequence similarity to SEQID NO:1 and a most preferred H-nm23 variant is one having at least 95%amino acid sequence similarity to SEQ ID NO:1.

The nucleic acid sequence encoding a portion of H-nm23 was firstidentified in the cDNA, Incyte Clone 964996, through acomputer-generated search for amino acid sequence alignments. Thenucleic acid sequence, SEQ ID NO:2, disclosed herein (FIGS. 1A and 1B)encodes the amino acid sequence, SEQ ID NO:1, designated H-nm23. Thefull length cDNA was assembled from Incyte Clones 603550 (BRSTTUT01);668015 (SCORNOT01); 669792 (CRBLNOT01); 964996 (BRSTNOT05); 1211471(BRSTNOT02); 1312720 (BLADTUT02); and 1403767 (LATRTUT02) from theLIFESEQ™ database (Incyte Pharmaceuticals, Palo Alto, Calif.).

The present invention is based in part on the structural homology shownin FIG. 2, among H-nm23 and other human nm23 isoforms including DR-nm23(GI 1051256; Venturelli D et al, supra), nm23-H1 (GI 468542, Rosengard AM et al, supra) and nm23-H2 (GI 127983, Stahl J A et al, supra).DR-nm23, nm23-H1, and nm23-H2 have, respectively, 50%, 46% and 44% aminoacid sequence identity to H-nm23.

The H-nm23 protein sequence consists of 187 amino acids. From positions68 to 97 the amino acid sequence contains a periodic repetition ofleucine residues at every sixth to seventh position. This pattern ischaracteristic of a leucine zipper motif, which is present in many generegulatory proteins (Busch S J et al (1990) Trends Genet 6:36-40).H-nm23 has an arg-gly-asp sequence at positions 138-140. This sequence,known as the "RGD motif", is crucial in the interaction of fibronectinwith its integrin cell-surface receptor, and plays a role in celladhesion (d'Souza S E et al (1991) Trends Biochem Sci 16:246-250).H-nm23 also contains the consensus sequence for NDPK (Gilles A M et al,supra) which includes the active site histidine at position 151.

THE H-nm23 CODING SEQUENCES

The nucleic acid and amino acid sequences of H-nm23 are shown in FIGS.1A and 1B. In accordance with the invention, any nucleic acid sequencewhich encodes the amino acid sequence of H-nm23 can be used to generaterecombinant molecules which express H-nm23. In a specific embodimentdescribed herein, a partial sequence of H-nm23 was first isolated asIncyte Clone 964996 from a human breast tissue library (BRSTNOT05).

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding H-nm23, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.The invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring H-nm23, and all such variations are tobe considered as being specifically disclosed.

Although nucleotide sequences which encode H-nm23 and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring H-nm23 under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding H-nm23 or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding H-nm23 and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

It is now possible to produce a DNA sequence, or portions thereof,encoding a H-mm23 and its derivatives entirely by synthetic chemistry,after which the synthetic gene may be inserted into any of the manyavailable DNA vectors and cell systems using reagents that are wellknown in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into agene encoding H-nm23.

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of FIGS. 1A and 1B under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.)incorporated herein by reference, and confer may be used at a definedstringency.

Altered nucleic acid sequences encoding H-nm23 which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent H-nm23. The proteinmay also show deletions, insertions or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent H-nm23. Deliberate amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of H-nm23 is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles ofH-nm23. As used herein, an "allele" or "allelic sequence" is analternative form of H-nm23. Alleles result from a mutation, ie, a changein the nucleic acid sequence, and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one or many allelic forms. Common mutationalchanges which give rise to alleles are generally ascribed to naturaldeletions, additions or substitutions of amino acids. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

Methods for DNA sequencing are well known in the art and employ suchenzymes as the Klenow fragment of DNA polymerase I, Sequenase® (USBiochemical Corp, Cleveland Ohio), Taq polymerase (Perkin Elmer, NorwalkConn.), thermostable T7 polymerase (Amersham, Chicago Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

EXTENDING THE POLYNUCLEOTIDE SEQUENCE

The polynucleotide sequence encoding H-nm23 may be extended utilizingpartial nucleotide sequence and various methods known in the art todetect upstream sequences such as promoters and regulatory elements.Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose"restriction-site" polymerase chain reaction (PCR) as a direct methodwhich uses universal primers to retrieve unknown sequence adjacent to aknown locus. First, genomic DNA is amplified in the presence of primerto a linker sequence and a primer specific to the known region. Theamplified sequences are subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergentprimers based on a known region (Triglia T et al (1988) Nucleic AcidsRes 16:8186). The primers may be designed using OLIGO® 4.06 PrimerAnalysis Software (1992; National Biosciences Inc, Plymouth Minn.), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1:111-19) is amethod for PCR amplification of DNA fragments adjacent to a knownsequence in human and yeast artificial chromosome DNA. Capture PCR alsorequires multiple restriction enzyme digestions and ligations to placean engineered double-stranded sequence into an unknown portion of theDNA molecule before PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker J D et al (1991; Nucleic Acids Res 19:3055-60). Additionally,one can use PCR, nested primers and PromoterFinder libraries to walk ingenomic DNA (PromoterFinder™ Clontech (Palo Alto Calif.). This processavoids the need to screen libraries and is useful in finding intron/exonjunctions.

Preferred libraries for screening for full length cDNAs are ones thathave been size-selected to include larger cDNAs. Also, random primedlibraries are preferred in that they will contain more sequences whichcontain the 5' and upstream regions of genes. A randomly primed librarymay be particularly useful if an oligo d(T) library does not yield afull-length cDNA. Genomic libraries are useful for extension into the 5'nontranslated regulatory region.

Capillary electrophoresis may be used to analyze the size or confirm thenucleotide sequence of sequencing or PCR products. Systems for rapidsequencing are available from Perkin Elmer, Beckman Instruments(Fullerton Calif.), and other companies. Capillary sequencing may employflowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity is converted to electrical signal usingappropriate software (eg. Genotyper™ and Sequence Navigator™ from PerkinElmer) and the entire process from loading of samples to computeranalysis and electronic data display is computer controlled. Capillaryelectrophoresis is particularly suited to the sequencing of small piecesof DNA which might be present in limited amounts in a particular sample.The reproducible sequencing of up to 350 bp of M13 phage DNA in 30 minhas been reported (Ruiz-Martinez M C et al (1993) Anal Chem 65:2851-8).

EXPRESSION OF THE NUCLEOTIDE SEQUENCE

In accordance with the present invention, polynucleotide sequences whichencode H-nm23, fragments of the polypeptide, fusion proteins orfunctional equivalents thereof may be used in recombinant DNA moleculesthe direct the expression of H-nm23 in appropriate host cells. Due tothe inherent degeneracy of the genetic code, other DNA sequences whichencode substantially the same or a functionally equivalent amino acidsequence, may be used to clone and express H-nm23. As will be understoodby those of skill in the art, it may be advantageous to produceH-nm23-encoding nucleotide sequences possessing non-naturally occurringcodons. Codons preferred by a particular prokaryotic or eukaryotic host(Murray E et al (1989) Nuc Acids Res 17:477-508) can be selected, forexample, to increase the rate of H-nm23 expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life, than transcripts produced from naturally occurringsequence.

The nucleotide sequences of the present invention can be engineered inorder to alter a coding sequence of H-nm23 for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, eg,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, etc.

In another embodiment of the invention, a natural, modified orrecombinant nucleotide sequence encoding H-nm23 may be ligated to aheterologous sequence to encode a fusion protein. For example, forscreening of peptide libraries for inhibitors of H-nm23 activity, it maybe useful to encode a chimeric H-nm23 protein that is recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between a H-nm23 sequence and theheterologous protein sequence, so that the H-nm23 may be cleaved andsubstantially purified away from the heterologous moiety.

In an alternate embodiment of the invention, the coding sequence forH-nm23 may be synthesized, whole or in part, using chemical methods wellknown in the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc).Alternatively, the protein itself could be produced using chemicalmethods to synthesize a H-nm23 amino acid sequence, whole or in part.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge J Y et al (1995) Science 269:202-204)and automated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (eg, Creighton (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co, NewYork N.Y.). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (eg, the Edman degradationprocedure; Creighton, supra). Additionally the amino acid sequence ofH-nm23, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with sequences from otherproteins, or any part thereof, to produce a variant polypeptide.

EXPRESSION SYSTEMS

In order to express a biologically active H-nm23, the nucleotidesequence encoding H-nm23 or its functional equivalent, is inserted intoan appropriate expression vector, ie, a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a H-nm23 coding sequence andappropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination or genetic recombination. Such techniques aredescribed in Sambrook et al (1989) Molecular Clonina, A LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y. and Ausubel F M et al(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y.

A variety of expression vector/host systems may be utilized to containand express a H-nm23 coding sequence. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (eg, baculovirus); plant cell systemstransfected with virus expression vectors (eg, cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.

The "control elements" or "regulatory sequences" of these systems varyin their strength and specificities and are those nontranslated regionsof the vector, enhancers, promoters, and 3' untranslated regions, whichinteract with host cellular proteins to carry out transcription andtranslation. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla Calif.)or pSport1 (Gibco BRL) and ptrp-lac hybrids and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (eg, heatshock, RUBISCO; and storage protein genes) or from plant viruses (eg,viral promoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from the mammalian genes or frommammalian viruses are most appropriate. If it is necessary to generate acell line that contains multiple copies of H-nm23, vectors based on SV40or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for H-nm23. For example, when largequantities of H-nm23 are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be desirable. Such vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the H-nm23 coding sequence may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster (1989) J BiolChem 264:5503-5509); and the like. pGEX vectors (Promega, Madison Wis.)may also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharcmyces cerevisiae, a number of vectors containingccnstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al (supra) andGrant et al (1987) Methods in Enzymology 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding H-nm23 may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMV(Brisson et al (1984) Nature 310:511-514) may be used alone or incombination with the omega leader sequence from TMV (Takamatsu et al(1987) EMBO J 6:307-311). Alternatively, plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3:1671-1680;Broglie et al (1984) Science 224:838-843); or heat shock promoters(Winter J and Sinibaldi R M (1991) Results Probl Cell Differ 17:85-105)may be used. These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. For reviewsof such techniques, see Hobbs S or Murry L E in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill New York N.Y., pp 191-196 orWeissbach and Weissbach (1988) Methods for Plant Molecular Biology,Academic Press, New York N.Y., pp 421-463.

An alternative expression system which could be used to express H-nm23is an insect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The H-nm23coding sequence may be cloned into a nonessential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the H-nm23 coding sequence will renderthe polyhedrin gene inactive and produce recombinant virus lacking coatprotein coat. The recombinant viruses are then used to infect S.frugiperda cells or Trichoplusia larvae in which H-nm23 is expressed(Smith et al (1983) J Virol 46:584; Engelhard E K et al (1994) Proc NatAcad Sci 91:3224-7).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence for H-nm23 may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingH-nm23 in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci81:3655-59). In addition, transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals may also be required for efficienttranslation of a H-nm23 sequence. These signals include the ATGinitiation codon and adjacent sequences. In cases where nucleic acidencoding H-nm23, its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate to the cell system inuse (Scharf D et al (1994) Results Probl Cell Differ 20:125-62; Bittneret al (1987) Methods in Enzymol 153:516-544).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressH-nm23 may be transformed using expression vectors which contain viralorigins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M et al (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) geneswhich can be employed in tk- or aprt- cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt,which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin F et al (1981) J Mol Biol 150:1-14) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman S C and R C Mulligan(1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins, βglucuronidase and its substrate, GUS, and luciferase and its substrate,luciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes C A et al (1995)Methods Mol Biol 55:121-131).

IDENTIFICATION OF TRANSFORMA TS CONTAINING THE POLYNUCLEOTIDE SEQUENCE

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expressionshould.be confirmed. For example, if the H-nm23 polynucleotide sequenceis inserted within a marker gene sequence, recombinant cells containingH-nm23 can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with a H-nm23sequence under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem H-nm23 as well.

Alternatively, host cells which contain the coding sequence for H-nm23and express H-nm23 may be dentified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA--DNA or DNA-RNA hybridization and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

The presence of the polynucleotide sequence encoding H-nm23 can bedetected by DNA--DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of H-nm23-encoding nucleotides. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the H-nm23 sequence to detect transformantscontaining H-nm23 DNA or RNA. As used herein "oligonucleotides" or"oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides which can beused as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofH-nm23, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson H-nm23 is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul Minn.) and Maddox D E et al (1983, J Exp Med 158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to H-nm23 include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the H-nm23 sequence, or any portion of it,may be cloned into a vector for the production of an mRNA probe. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmac.a Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminscent, or chromogenic agents as well as substrates, cofactors,inhibitors, magnetic particles and the like. Patents teaching the use ofsuch labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinantimmunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567incorporated herein by reference.

PURIFICATION OF H-nm23

Host cells transformed with a H-nm23-encoding nucleotide sequence may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell may be contained intracellularly or secreted dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing H-nm23 can bedesigned for efficient production and proper transmembrane insertion ofH-nm23 into a prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may join H-nm23 to nucleotide sequence encoding apolypeptide domain which will facilitate purification of solubleproteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53; cf discussionof vectors infra containing fusion proteins).

H-nm23 may also be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequences such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and H-nm23 is useful to facilitate purification. One suchexpression vector provides for expression of a fusion proteincompromising an H-nm23 and contains nucleic acid encoding 6 histidineresidues followed by thioredoxin and an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography as described in Porath et al (1992) ProteinExpression and Purification 3: 263-281) while the enterokinase cleavagesite provides a means for purifying the H-nm23 from the fusion protein.

In addition to recombinant production, fragments of H-nm23 may beproduced by direct peptide synthesis using solid-phase techniques (cfStewart et al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, SanFrancisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster CityCalif.) in accordance with the instructions provided by themanufacturer. Various fragments of H-nm23 may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

USES OF H-nm23

The rationale for the use of polynucleotide and polypeptide sequencesdisclosed herein is based in part on the chemical and structuralhomology among the novel H-nm23 and human isoforms of nm23. H-nm23 maybe used in the diagnosis, prognosis and treatment of diseases associatedwith abnormal tissue development and differentiation including cancer.

H-nm23 may be useful as a tumor suppressor or a metastatic inhibitor,decreasing the severity or the metastatic potential of certain tumortypes.

A correlation between expression of H-nm23 and metastasis in specifictumors would provide a measure of metastatic potential. In such tumortypes, H-nm23 may be useful as a prognostic marker. Antibodiesspecifically recognizing H-nm23 may be used to quantitate H-nm23 forprognostic and diagnostic purposes.

H-nm23 or its fragments can be used to identify specific molecules withwhich it interacts. Such molecules include agonists that enhance H-nm23activity. Furthermore, DNA segments to which H-nm23 binds may revealpromoter regions or other regulatory sites important in the control ofcell cycle or tumor-associated genes.

H-nm23 ANTIBODIES

H-nm23-specific antibodies are useful for the diagnosis and prognosis ofconditions and diseases associated with expression of H-nm23. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library. Neutralizing antibodies, ie, those which inhibitdimer formation, are especially preferred for diagnostics andtherapeutics.

H-nm23 for antibody induction does not require biological activity;however, the protein fragment, or oligopeptide must be antigenic.Peptides used to induce specific antibodies may have an amino acidsequence consisting of at least five amino acids, preferably at least 10amino acids. Preferably, they should mimic a portion of the amino acidsequence of the natural protein and may contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofH-nm23 amino acids may be fused with those of another protein such askeyhole limpet hemocyanin and antibody produced against the chimericmolecule. Procedures well known in the art can be used for theproduction of antibodies to H-nm23.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with H-nm23 orany portion, fragment or oligopeptide which retains immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium paryumare potentially useful human adjuvants.

Monoclonal antibodies to H-nm23 may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Koehler and Milstein (1975Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al(1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985)Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal 1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce H-nm23-specific single chain antibodies

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for H-nm23 mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al (1989) Science 256:1275-1281).

A variety of protocols for competitive binding or immunoradiometricassays using either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the formation of complexes between H-nm23 and its specificantibody and the measurement of complex formation. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo noninterfering epitopes on a specific H-nm23 protein is preferred,but a competitive binding assay may also be employed. These assays aredescribed in Maddox D E et al (1983, J Exp Med 158:1211).

DIAGNOSTIC ASSAYS USING H-nm23 SPECIFIC ANTIBODIES

Particular H-nm23 antibodies are useful for the diagnosis of conditionsor diseases characterized by expression of H-nm23 or in assays tomonitor patients being treated with H-nm23, agonists or inhibitors.Diagnostic assays for H-nm23 include methods utilizing the antibody anda label to detect H-nm23 in human body fluids or extracts of cells ortissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a reporter molecule. A wide variety of reportermolecules are known, several of which were described above.

A variety of protocols for measuring H-nm23, using either polyclonal ormonoclonal antibodies specific for the respective protein are known inthe art. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on H-nm23 is preferred, but acompetitive binding assay may be employed. These assays are described,among other places, in Maddox, D E et al (1983, J Exp Med 158:1211).

In order to provide a basis for diagnosis, normal or standard values forH-nm23 expression must be established. This is accomplished by combiningbody fluids or cell extracts taken from normal subjects, either animalor human, with antibody to H-nm23 under conditions suitable for complexformation which are well known in the art. The amount of standardcomplex formation may be quantified by comparing various artificialmembranes containing known quantities of H-nm23 with both control anddisease samples from biopsied tissues. Then, standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom subjects potentially affected by disease. Deviation betweenstandard and subject values establishes the presence of disease state.

DRUG SCREENING

H-nm23, its catalytic or immunogenic fragments or oligopeptides thereof,can be used for screening therapeutic compounds in any of a variety ofdrug screening techniques. The fragment employed in such a test may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenH-nm23 and the agent being tested, may be measured.

Another technique for drug screening which may be used for highthroughput screening of compounds having suitable binding affinity tothe H-nm23 is described in detail in "Determination of Amino AcidSequence Antigenicity" by Geysen H M, WO Application 84/03564, publishedon Sep. 13, 1984, and incorporated herein by reference. In summary,large numbers of different small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are reacted with fragments of H-nm23 and washed.Bound H-nm23 is then detected by methods well known in the art.Substantially purified H-nm23 can also be coated directly onto platesfor use in the aforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding H-nm23specifically compete with a test compound for binding H-nm23. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with H-nm23.

USES OF THE POLYNUCLEOTIDE ENCODING H-nm23

A polynucleotide encoding H-nm23, or any part thereof, may be used fordiagnostic and/or therapeutic purposes. For diagnostic purposes, theH-nm23 of this invention may be used to detect and quantitate geneexpression in biopsied tissues in which expression of H-nm23 may beimplicated. The diagnostic assay is useful to distinguish betweenabsence, presence, and excess expression of H-nm23 and to monitorregulation of H-nm23 levels during therapeutic intervention. Included inthe scope of the invention are oligonucleotide sequences, antisense RNAand DNA molecules, and PNAS.

Another aspect of the subject invention is to provide for hybridizationor PCR probes which are capable of detecting polynucleotide sequences,including genomic sequences, encoding H-nm23 or closely relatedmolecules. The specificity of the probe, whether it is made from ahighly specific region, eg, 10 unique nucleotides in the 5' regulatoryregion, or a less specific region, eg, especially in the 3' region, andthe stringency of the hybridization or amplification (maximal, high,intermediate or low) will determine whether the probe identifies onlynaturally occurring H-nm23, alleles or related sequences.

Probes may also be used for the detection of related sequences andshould preferably contain at least 50% of the nucleotides from any ofthese H-nm23 encoding sequences. The hybridization probes of the subjectinvention may be derived from the nucleotide sequence of SEQ ID NO:2 orfrom genomic sequence including promoter, enhancer elements and intronsof the naturally occurring H-nm23. Hybridization probes may be labeledby a variety of reporter groups, including radionuclides such as ³² P or³⁵ S, or enzymatic labels such as alkaline phosphatase coupled to theprobe via avidin/biotin coupling systems, and the like.

Other means for producing specific hybridization probes for H-nm23 DNAsinclude the cloning of nucleic acid sequences encoding H-nm23 or H-nm23derivatives into vectors for the production of mRNA probes. Such vectorsare known in the art and are commercially available and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate radioactively labeled nucleotides.

Diagnostic Use

Polynucleotide sequences encoding H-nm23 may be used for the diagnosisof conditions or diseases with which the expression of H-nm23 isassociated. For example, polynucleotide sequences encoding H-nm23 may beused in hybridization or PCR assays of fluids or tissues from biopsiesto detect H-nm23 expression. The form of such qualitative orquantitative methods may include Southern or northern analysis, dot blotor other membrane-based technologies; PCR technologies; dip stick, pin,chip and ELISA technologies. All of these techniques are well known inthe art and are the basis of many commercially available diagnostickits.

The H-nm23 nucleotide sequence disclosed herein provide the basis forassays that detect activation or induction associated with disease. TheH-nm23 nucleotide sequence may be labeled by methods known in the artand added to a fluid or tissue sample from a patient under conditionssuitable for the formation of hybridization complexes. After anincubation period, the sample is washed with a compatible fluid whichoptionally contains a dye (or other label requiring a developer) if thenucleotide has been labeled with an enzyme. After the compatible fluidis rinsed off, the dye is quantitated and compared with a standard. Ifthe amount of dye in the biopsied or extracted sample is significantlyelevated over that of a comparable control sample, the nucleotidesequence has hybridized with nucleotide sequences in the sample, and thepresence of elevated levels of H-nm23 nucleotide sequences in the sampleindicates the presence of the associated inflammation and/or disease.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regime in animal studies, in clinical trials, orin monitoring the treatment of an individual patient. In order toprovide a basis for the diagnosis of disease, a normal or standardprofile for H-nm23 expression must be established. This is accomplishedby combining body fluids or cell extracts taken from normal subjects,either animal or human, with H-nm23, or a portion thereof, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fornormal subjects with a dilution series of H-nm23 run in the sameexperiment where a known amount of substantially purified H-nm23 isused. Standard values obtained from normal samples may be compared withvalues obtained from samples from patients afflicted withH-nm23--associated diseases. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established, a therapeutic agent is administered and atreatment profile is generated. Such assays may be repeated on a regularbasis to evaluate whether the values in the profile progress toward orreturn to the normal or standard pattern. Successive treatment profilesmay be used to show the efficacy of treatment over a period of severaldays or several months.

Polymerase Chain Reaction (PCR) as described in U.S. Pat. Nos. 4,683,195and 4,965,188 provides additional uses for oligonucleotides based uponthe H-nm23 sequence. Such oligomers are generally chemicallysynthesized, but they may be generated enzymatically or produced from arecombinant source. Oligomers generally comprise two nucleotidesequences, one with sense orientation (5'→3') and one with antisense(3'←5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Additionally, methods which may be used to quantitate the expression ofa particular molecule include radiolabeling (Melby PC et al 1993 JImmunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 AnalBiochem 229-36) nucleotides, coamplification of a control nucleic acid,and standard curves onto which the experimental results areinterpolated. Quantitation of multiple samples may be speeded up byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation. A definitive diagnosis of this typemay allow health professionals to begin aggressive treatment and preventfurther worsening of the condition. Similarly, further assays can beused to monitor the progress of a patient during treatment. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown such as the triplet genetic code, specific base pair interactions,and the like.

Therapeutic Use

Based upon its homology to the genes encoding the nm23 isoforms and itsexpression profile, the H-nm23 polynucleotide disclosed herein mayprovide the basis for the design of molecules for the treatment ofcancer or other disorders associated with abnormal tissue development ordifferentiation.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisenseH-nm23. See, for example, the techniques described in Sambrook et al(supra) and Ausubel et al (supra).

The polynucleotides comprising full length cDNA sequence and/or itsregulatory elements enable researchers to use H-nm23 as an investigativetool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem60:631-652) regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers, or larger fragments,can be designed from various locations along the coding or controlregions.

Genes encoding H-nm23 can be turned off by transfecting a cell or tissuewith expression vectors which express high levels of a desired H-nm23fragment. Such constructs can flood cells with untranslatable sense orantisense sequences. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until all copiesare disabled by endogenous nucleases. Transient expression may last fora month or more with a non-replicating vector (Mettler I, personalcommunication) and even longer if appropriate replication elements arepart of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA or PNA, to the control regionsof H-nm23, ie, the promoters, enhancers, and introns. Olgonucleotidesderived from the transcription initiation site, eg, between -10 and +10regions of the leader sequence, are preferred. The antisense moleculesmay also be designed to block translation of mRNA by preventing thetranscript from binding to ribosomes. Similarly, inhibition can beachieved using "triple helix" base-pairing methodology. Triple helixpairing compromises the ability of the double helix to open sufficientlyfor the binding of polymerases, transcription factors, or regulatorymolecules. Recent therapeutic advances using triplex DNA were reviewedby Gee J E et al (In: Huber B E and B I Carr (1994) Molecular andImmunoloaic Approaches, Futura Publishing Co, Mt Kisco N.Y.).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of RNA encoding H-nm23.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding H-nm23. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutnvely or inducibly can be introduced into celllines, cells or tissues.

RNA molecules may be modified to in crease intracellular stability andhalf-life . Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanpheosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Methods for introducing vectors into cells or tissues include thosemethods discussed infra and which are equally suitable for in vivo, invitro and ex vivo therapy. For ex vivo therapy, vectors are introducedinto stem cells taken from the patient and clonally propagated forautologous transplant back into that same patient is presented in U.S.Pat. Nos. 5,399,493 and 5,437,994, disclosed herein by reference.Delivery by transfection and by liposome are quite well known in theart.

Furthermore, the nucleotide sequences for H-nm23 disclosed herein may beused in molecular biology techniques that have not yet been developed,provided the new techniques rely on properties of nucleotide sequencesthat are currently known, including but not limited to such propertiesas the triplet genetic code and specific base pair interactions.

DETECTION AND MAPPING OF RELATED POLYNUCLEOTIDE SEQUENCES

The nucleic acid sequence for H-nm23 can also be used to generatehybridization probes for mapping the naturally occurring genomicsequence. The sequence may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Theseinclude in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price C M (1993; Blood Rev 7:127-34) and Trask B J 1991;Trends Genet 7:149-54).

The technique of fluorescent in situ hybridization of chromosome spreadshas been described, among other places, in Verma et al (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofa H-nm23 on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. For example, an STSbased map of the human genome was recently published by theWhitehead-MIT Center for Genomic Research (Hudson T J et al (1995)Science 270:1945-1954). Often the placement of a gene on the chromosomeof another mammalian species such as mouse (Whitehead Institute/MITCenter for Genome Research, Genetic Map of the Mouse, Database Release10, Apr. 28, 1995) may reveal associated markers even if the number orarm of a particular human chromosome is not known. New sequences can beassigned to chromosomal arms, or parts thereof, by physical mapping.This provides valuable information to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome, such as ataxia telangiectasia(AT), has been crudely localized by genetic linkage to a particulargenomic region, for example, AT to 11q22-23 (Gatti et al (1988) Nature336:577-580); any sequences mapping to that area may representassociated or regulatory genes for further investigation. The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc. amongnormal, carrier or affected individuals.

PHARMACEUTICAL COMPOSITIONS

The present invention relates to pharmaceutical compositions which maycomprise nucleotides, proteins, antibodies, agonists, antagonists, orinhibitors, alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. Any of these molecules canbe administered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In one embodimentof the present invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

Administration of Pharmaceutical Compositions

Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co,Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie, dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used n the formulation. Suchpenetrants are generally known n the art.

Manufacture and Storage

The pharmaceutical compositions of the present invention may bemanufactured in a manner that known in the art, eg, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of H-nm23, such labeling wouldinclude amount, frequency and method of administration.

Therapeutically Effective Dose

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, eg, of neoplastic cells, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of protein or itsantibodies, antagonists, or inhibitors which ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, eg, ED50 (the dose therapeutically effective in50% of the population) and LD50 the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, eg, tumor size and location; age, weightand gender of the patient; diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

The normal breast (BRSNOT05) and breast carcinoma (BRSTTUT03) cDNAlibraries were constructed from the breast tissue of a 58 year oldCaucasian female. The patient was diagnosed with multicentric invasivegrade 4 lobular carcinoma. The normal breast tissue is adjacent,microscopically normal tissue. (specimens #0116A and #0116B; MayoClinic, Rochester Minn.).

The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.0,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water and DNase treated at 37° C. The RNAextraction was repeated with acid phenol chloroform pH 8.0 andprecipitated with sodium acetate and ethanol as before. The mRNA wasthen isolated using the Qiagen Oligotex kit (QIAGEN Inc; ChatsworthCalif.) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSuperscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013; Gibco/BRL), cDNAs were fractionated on a Sepharose CL4Bcolumn (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pSport I. The plasmid pSport I was subsequentlytransformed into DH5a™ competent cells (Cat. #18258-012; Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit for Rapid Extraction Alkaline Lysis Plasmid Minipreps(Catalog #26173; QIAGEN, Inc). This kit enables the simultaneouspurification of 96 samples in a 96-well block using multi-channelreagent dispensers. The recommended protocol was employed except for thefollowing changes: 1) the bacteria were cultured in 1 ml of sterileTerrific Broth (Catalog #22711, LIFE TECHNOLOGIES™) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 36-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger F and A R Coulson(1375; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno Nev.) in combination with Peltier Thermal Cyclers (PTC200 from MJResearch, Watertown Mass.) and Applied Biosystems 377 DNA SequencingSystems; and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT-670Sequence Analysis System. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT™ 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul S F(1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol215:403-10), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al. supra).

Analogous computer techniques use BLAST (Altschul S F 1993 and 1990,supra) to search for identical or related molecules in nucleotidedatabases such as GenBank or the LIFESEQ™ database (Incyte, Palo AltoCalif.). This analysis is much faster than multiple, membrane-basedhybridizations. In addition, the sensitivity of the computer search canbe modified to determine whether any particular match is categorized asexact or homologous.

The basis of the search is the product score which is defined as:##EQU1## and it takes into account both the degree of similarity betweentwo sequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

The results of the search are reported as a list of libraries in whichthe full length sequence, or parts thereof, is represented, theabundance of the sequence, and the percent abundance. Abundance directlyreflects the number of times a particular transcript is present in acDNA library, and percent abundance is abundance divided by the totalnumber of sequences examined in the library.

V Extension of H-nm23 to Full Length or to Recover Regulatory Elements

The nucleic acid sequence encoding full length H-nm23 (SEQ ID NO:2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5' sequences fromgenomic libraries. One primer is synthesized to initiate extension inthe antisense direction (XLR) and the other is synthesized to extendsequence in the sense direction (XLF). Primers allow the extension ofthe known H-nm23 nucleotide sequence "outward" generating ampliconscontaining new, unknown nucleotide sequence for the region of interest(U.S. patent application Ser. No. 08/487,112, filed Jun. 7, 1995,specifically incorporated by reference). The initial primers aredesigned from the cDNA using OLIGO® 4.06 Primer Analysis Software(National Biosciences), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; MJ Research,Watertown Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                     Step 2 65° C. for 1 min                                                Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products were selected and cut out of the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc). After recovery of the DNA, Klenow enzymewas used to trim single-stranded, nucleotide overhangs creating bluntends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic ONAs or mRNAs. Although the labeling of oligonucleotides,consisting of about 20 base-pairs, is specifically described,essentially the same procedure is used with larger cDNA fragments.Oligonucleotides are designed using state-of-the-art software such asOLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of eacholigomer and 250 mCi of [γ-³² P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 super fine resin column (Pharmacia). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale Calif.) for several hours,hybridization patterns are compared visually.

VII Antisonse Molecules

The nucleotide sequence encoding H-nm23, or any part thereof, is used toinhibit in vivo or in vitro expression of naturally occurring H-nm23.Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequence of H-nm23 as shown in FIGS. 1A and 1B is used to inhibitexpression of naturally occurring H-nm23. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an H-nm23 transcript by preventing the ribosome from binding. Usingan appropriate portion of the leader and 5' sequence of SEQ ID NO:2, aneffective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or early codingsequence of the polypeptide as shown in FIGS. 1A and 1B.

VIII Expression of H-nm23

Expression of H-nm23 is accomplished by subcloning the cDNAs intoappropriate vectors and transfecting the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express H-nm23 in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Metand the subsequent 7 residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transfected bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firstseven residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length H-nm23. The signal sequence directs the secretion ofH-nm23 into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX H-nm23 Activity

Binding of H-nm23 to DNA is assayed by monitoring the differences inelectrophoretic mobility of the DNA with and without protein bound. ADNA probe (for example, a 105 base pair double-stranded fragmentcomprising the c-myc promoter region) is radiolabeled with ³² P by thetransfer of the terminal phosphate of [γ-³² P]ATP to the 5' ends of theDNA, catalyzed by T4 polynucleotide kinase (New England Biolabs, BeverlyMass.). The radiolabeled DNA is incubated with varying concentrations ofH-nm23 at room temperature for approximately 1 hour. The incubationreactions are electrophoresed through non-denaturing polyacrylamidegels. The gels are dried and autoradiographed. The relative intensitiesof the bands corresponding to uncomplexed DNA and protein-complexed DNAfor each concentration of added H-nm23 are determined by densitometery.The resulting tinration curve is used along with the concentrations ofthe radiolabeled DNA and added H-nm23 to calculate values for theaffinity of H-nm23 for the DNA.

The NDPK activity of H-nm23 is measured in a coupled pyruvatekinase-lactate dehydrogenase assay system (Sigma Chemical Co., St. LouisMo.), with ATP as the phosphate donor and dTDP as the phosphate acceptornucleotides. The ADP produced in the NDPK reaction reacts withphosphoenolpyruvate via the pyruvate kinase to form pyruvate. Tn thepresence of pyruvate, the lactate dehydrogenase oxidizes NADH. NADHoxidation is accompanied by a change in absorbance at 340 nm, which ismonitored in a spectrophotometer.

X Production of H-nm23 Specific Antibodies

H-nm23 substantially purified using PAGE electrophoresis (Sambrook,supra) is used to immunize rabbits and to produce antibodies usingstandard protocols. The amino acid sequence translated from H-nm23 isanalyzed using DNAStar software (DNAStar Inc) to determine regions ofhigh immunogenicity and a corresponding oligopolypeptide is synthesizedand used to raise antibodies by means known to those of skill in theart. Analysis to select appropriate epitopes, such as those near theC-terminus or in hydrophilic regions (shown in FIG. 3) is described byAusubel F M et al (supra).

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) byreaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS;Ausubel F M et al, supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring H-nm23 Using Specific Antibodies

Naturally occurring or recombinant H-nrn23 is substantially purified byimmunoaffinity chromatograpny using antibodies specific for H-nm23. Animmunoaffinity column is constructed by covalently coupling H-nm23antircy to an activated chromatographic resin such as CnBr-activatedSepharose (Pharmacia Biotech). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Cellular fractions from cells containing H-nm23 are prepared bysolubilization of the whole cell and isolation of subcellular fractionsby differential centrifugation, by the addition of detergent, or byother methods well known in the art. Alternatively, soluble H-nm23containing a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A fractionated H-nm23-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of H-nm23 (eg, high ionic strengthbuffers in the presence of detergent). The column is eluted underconditions that disrupt antibody/H-nm23 binding (eg, a buffer of pH 2-3or a high concentration of a chaotrope such as urea or thiocyanate ion),and H-nm23 is collected.

XII Identification of Molecules Which Interact with H-nm23

H-nm23 is useful as a research tools for identification,characterization and purification of molecules with which it interacts.In one embodiment of affinity purification, H-nm23 is covalently coupledto a chromatography column. Cells and their membranes are extracted,endogenous H-nm23 is removed and various H-nm23-free subcomponents arepassed over the column. H-nm23-associated molecules bind to the columnby virtue of their biological affinity. The H-nm23-complex is recoveredfrom the column, dissociated and the recovered molecule is subjected toN-terminal protein sequencing, nucleic acid sequencing, orhigh-performance liquid chromatography/mass spectrometry (HPLC/MS),depending on the type of molecule. The amino acid or nucleotide sequenceor mass spectral analysis is then used to identify the captured moleculeor, in the case of a protein ligand, to design degenerateoligonucleotide probes for cloning its gene from an appropriate cDNAlibrary.

In an alternate method, monoclonal antibodies are raised against H-nm23and screened to identify those compounds which inhibit the binding ofthe antibody to H-nm23. These monoclonal antibodies may then used inaffinity purification or expression cloning of associated molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 5                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 187 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY:                                                                  (B) CLONE: Consensus                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Gly Gly Leu Phe Trp Arg Ser Ala Leu - #Arg Gly Leu Arg Cys        Gly                                                                               1               5 - #                 10 - #                 15             - -  Pro Arg Ala Pro Gly Pro Ser Leu Leu Val - #Arg His Gly Ser Gly Gly                   20     - #             25     - #             30                  - -  Pro Ser Trp Thr Arg Glu Arg Thr Leu Val - #Ala Val Lys Pro Asp Gly               35         - #         40         - #         45                      - -  Val Gln Arg Arg Leu Val Gly Asp Val Ile - #Gln Arg Phe Glu Arg Arg           50             - #     55             - #     60                          - -  Gly Phe Thr Leu Val Gly Met Lys Met Leu - #Gln Ala Pro Glu Ser Val       65                 - # 70                 - # 75                 - # 80       - -  Leu Ala Glu His Tyr Gln Asp Leu Arg Arg - #Lys Pro Phe Tyr Pro Ala                       85 - #                 90 - #                 95              - -  Leu Ile Arg Tyr Met Ser Ser Gly Pro Val - #Val Ala Met Val Trp Glu                   100     - #            105     - #            110                 - -  Gly Tyr Asn Val Val Arg Ala Ser Arg Ala - #Met Ile Gly His Thr Asp               115         - #        120         - #        125                     - -  Ser Ala Glu Ala Ala Pro Gly Thr Ile Arg - #Gly Asp Phe Ser Val His           130             - #    135             - #    140                         - -  Ile Ser Arg Asn Val Ile His Ala Ser Asp - #Ser Val Glu Gly Ala Gln       145                 - #150                 - #155                 -         #160                                                                             - -  Arg Glu Ile Gln Leu Trp Phe Gln Ser Ser - #Glu Leu Val Ser Trp        Ala                                                                                              165 - #                170 - #                175            - -  Asp Gly Gly Gln His Ser Ser Ile His Pro - #Ala                                       180     - #            185                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 986 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY:                                                                  (B) CLONE: Consensus                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  GGCCGGGCGT CATGGGCGGC CTCTTCTGGC GCTCCGCGCT GCGGGGGCTG - #CGCTGCGGC    C    60                                                                         - -  CGCGGGCCCC GGGCCCGAGC CTGCTAGTGC GCCACGGCTC GGGAGGGCCC - #TCCTGGACC    C   120                                                                         - -  GGGAGCGGAC CCTGGTGGCG GTGAAGCCCG ATGGCGTGCA ACGGCGGCTC - #GTTGGGGAC    G   180                                                                         - -  TGATCCAGCG CTTTGAGAGG CGGGGCTTCA CGCTGGTGGG GATGAAGATG - #CTGCAAGCA    C   240                                                                         - -  CAGAGAGCGT CCTTGCCGAG CACTACCAGG ACCTGCGGAG GAAGCCCTTC - #TACCCTGCC    C   300                                                                         - -  TMATCCGCTA CATGAGCTCT GGGCCTGTGG TGGCCATGGT CTGGGAAGGG - #TACAATGTC    G   360                                                                         - -  TCCGCGCCTC RAGGGCCATG ATTGGACACA CCGACTCGGC TGAGGCTGCC - #CCAGGAACC    A   420                                                                         - -  TAAGGGGTGA CTTCAGCGTC CACATCAGCA GGAATGTCAT CCACGCCAGC - #GACTCCGTG    G   480                                                                         - -  AGGGGGCCCA GCGGGAGATC CAGCTGTGGT TCCAGAGCAG TGAGCTGGTG - #AGCTGGGCA    G   540                                                                         - -  ACGGGGGCCA GCACAGCAGC ATCCACCCAG CCTGAGGCTC AAGCTGCCCT - #TACCACCCC    A   600                                                                         - -  TCCCCCACGC AGGACCAACT ACCTCCGTCA GCAAGAACCC AAGCCCACAT - #CCAAACCTG    C   660                                                                         - -  CTGTCCCAAA CCACTTACTT CCCTGTTCAC CTCTGCCCCA CCCCAGCCCA - #GAGGAGTTT    G   720                                                                         - -  AGCCACCAAC TTCAGTGCCT TTCTGTACCC CAAGCCAGCA CAAGATTGGA - #CCAATCCTT    T   780                                                                         - -  TTGCACCAAA GTGCCGGACA ACCTTTGTGG TGGGGGGGGG TCTTCACATT - #ATCATAACC    T   840                                                                         - -  CTCCTCTAAA GGGGAGGCAT TAAAATTCAC TGTGCCCAGC ACATGGGTGG - #TACACTAAT    T   900                                                                         - -  ATGACTTCCC CCAGCTCTGA GGTAGAAATG ACGCCTTTAT GCAAGTTGTA - #AGGAGTTGA    A   960                                                                         - -  CAGTAAAGAG GAAGTTTTGC ACACCC         - #                  - #                 986                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1051256                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Ile Cys Leu Val Leu Thr Ile Phe Ala - #Asn Leu Phe Pro Ala Ala        1               5 - #                 10 - #                 15              - -  Cys Thr Gly Ala His Glu Arg Thr Phe Leu - #Ala Val Lys Pro Asp Gly                   20     - #             25     - #             30                  - -  Val Gln Arg Arg Leu Val Gly Glu Ile Val - #Arg Arg Phe Glu Arg Lys               35         - #         40         - #         45                      - -  Gly Phe Lys Leu Val Ala Leu Lys Leu Val - #Gln Ser Ser Glu Glu Leu           50             - #     55             - #     60                          - -  Leu Arg Glu His Tyr Ala Glu Leu Arg Glu - #Arg Pro Phe Tyr Gly Arg       65                 - # 70                 - # 75                 - # 80       - -  Leu Val Lys Tyr Met Ala Ser Gly Pro Val - #Val Ala Met Val Trp Gln                       85 - #                 90 - #                 95              - -  Gly Leu Asp Val Val Arg Thr Ser Arg Ala - #Leu Ile Gly Ala Thr Asn                   100     - #            105     - #            110                 - -  Pro Ala Asp Ala Pro Pro Gly Thr Ile Arg - #Gly Asp Phe Cys Ile Glu               115         - #        120         - #        125                     - -  Val Gly Asn Leu Ile His Gly Ser Asp Ser - #Val Glu Ser Ala Arg Arg           130             - #    135             - #    140                         - -  Glu Ile Ala Leu Trp Phe Arg Ala Asp Glu - #Leu Leu Cys Trp Glu Asp       145                 - #150                 - #155                 -         #160                                                                             - -  Ser Ala Gly His Trp Leu Tyr Glu                                                          165                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 468542                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Met Ala Asn Cys Glu Arg Thr Phe Ile Ala - #Ile Lys Pro Asp Gly        Val                                                                               1               5 - #                 10 - #                 15             - -  Gln Arg Gly Leu Val Gly Glu Ile Ile Lys - #Arg Phe Glu Gln Lys Gly                   20     - #             25     - #             30                  - -  Phe Arg Leu Val Gly Leu Lys Phe Met Gln - #Ala Ser Glu Asp Leu Leu               35         - #         40         - #         45                      - -  Lys Glu His Tyr Val Asp Leu Lys Asp Arg - #Pro Phe Phe Ala Gly Leu           50             - #     55             - #     60                          - -  Val Lys Tyr Met His Ser Gly Pro Val Val - #Ala Met Val Trp Glu Gly       65                 - # 70                 - # 75                 - # 80       - -  Leu Asn Val Val Lys Thr Gly Arg Val Met - #Leu Gly Glu Thr Asn Pro                       85 - #                 90 - #                 95              - -  Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly - #Asp Phe Cys Ile Gln Val                   100     - #            105     - #            110                 - -  Gly Arg Asn Ile Ile His Gly Ser Asp Ser - #Val Glu Ser Ala Glu Lys               115         - #        120         - #        125                     - -  Glu Ile Gly Leu Trp Phe His Pro Glu Glu - #Leu Val Asp Tyr Thr Ser           130             - #    135             - #    140                         - -  Cys Ala Gln Asn Trp Ile Tyr Glu                                          145                 - #150                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 127983                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  Met Ala Asn Leu Glu Arg Thr Phe Ile Ala - #Ile Lys Pro Asp Gly Val        1               5 - #                 10 - #                 15              - -  Gln Arg Gly Leu Val Gly Glu Ile Ile Lys - #Arg Phe Glu Gln Lys Gly                   20     - #             25     - #             30                  - -  Phe Arg Leu Val Ala Met Lys Phe Leu Arg - #Ala Ser Glu Glu His Leu               35         - #         40         - #         45                      - -  Lys Gln His Tyr Ile Asp Leu Lys Asp Arg - #Pro Phe Phe Pro Gly Leu           50             - #     55             - #     60                          - -  Val Lys Tyr Met Asn Ser Gly Pro Val Val - #Ala Met Val Trp Glu Gly       65                 - # 70                 - # 75                 - # 80       - -  Leu Asn Val Val Lys Thr Gly Arg Val Met - #Leu Gly Glu Thr Asn Pro                       85 - #                 90 - #                 95              - -  Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly - #Asp Phe Cys Ile Gln Val                   100     - #            105     - #            110                 - -  Gly Arg Asn Ile Ile His Gly Ser Asp Ser - #Val Lys Ser Ala Glu Lys               115         - #        120         - #        125                     - -  Glu Ile Ser Leu Trp Phe Lys Pro Glu Glu - #Leu Val Asp Tyr Lys Ser           130             - #    135             - #    140                         - -  Cys Ala His Asp Trp Val Tyr Glu                                          145                 - #150                                                  __________________________________________________________________________

We claim:
 1. An isolated and purified polynucleotide encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:1.
 2. Amethod for producing the polypeptide of SEQ ID NO:1, the methodcomprising the steps of:a) culturing a host cell containing anexpression vector comprising a polynucleotide encoding the polypeptideof claim 1 under conditions suitable for expression of the polypeptide;and b) recovering the polypeptide from the host cell culture.